Identification of novel monocistronic HTLV-1 mRNAs encoding functional Rex isoforms
© Rende et al. 2015
Received: 20 January 2015
Accepted: 19 June 2015
Published: 2 July 2015
Human T cell leukemia virus type 1 (HTLV-1) gene expression is controlled by the key regulatory proteins Tax and Rex. The concerted action of these proteins results in a two-phase kinetics of viral expression that depends on a time delay between their action. However, it is difficult to explain this delay, as Tax and Rex are produced from the same mRNA. In the present study we investigated whether HTLV-1 may produce novel mRNA species capable of expressing Rex and Tax independently.
Results revealed the expression of three alternatively spliced transcripts coding for novel Rex isoforms in infected cell lines and in primary samples from infected patients. One mRNA coded for a Tax isoform and a Rex isoform, and two mRNAs coded for Rex isoforms but not Tax. Functional assays showed that these Rex isoforms exhibit activity comparable to canonic Rex. An analysis of the temporal expression of these transcripts upon ex vivo culture of cells from infected patients and cell lines transfected with a molecular clone of HTLV-1 revealed early expression of the dicistronic tax/rex mRNAs followed by the monocistronic mRNAs coding for Rex isoforms.
The production of monocistronic HTLV-1 mRNAs encoding Rex isoforms with comparable activity to canonical Rex, but with distinct timing, would support a prolonged duration of Rex function with gradual loss of Tax, and is consistent with the two-phase expression kinetics. A thorough understanding of these regulatory circuits will shed light on the basis of viral latency and provide groundwork to develop strategies for eradicating persistent infections.
Identification and coding potential of novel alternatively spliced HTLV-1 mRNAs
RT-PCR and qPCR primers and probes
RT-PCR and real time PCR primers
TaxRex s: 5′-GTCCGCCGTCTAG^CTTCC-3′ (exon 1^2 SA)
TaxRex as: 5′-CTGGGAAGTGGG^CCATGG-3′ (exon 2^3 SA)
TaxaRexa s: 5′-ACCACCAACACCATGG^GGTTTG-3′ (exon 2^3a SA)
Rexb s: 5′-ACCACCAACACCATGG^CAGGTC-3′ (exon 2^C SA)
Rexc s: 5′-ACCACCAACACCATGG^GTCCTC-3′ (exon 2^Ca SA)
Rex isoforms as: 5′-GAGTCGAGGGATAAGGAAC-3′
Real time probes
TaxRex: 5′ (FAM)-CCCAGTGGATCCCGTGGAG-3′(TAMRA)
Rex isoforms: 5′(FAM)-AAGGCGACTGGTGCCCCATCTCTGGG-3′(TAMRA)
Quantitation of novel alternatively spliced HTLV-1 mRNAs
Intracellular localization of Rex protein isoforms
Functional analysis of Rex protein isoforms
The finding that Rexb and Rexc show a functional activity comparable to that of Rex and Rexa suggests that their prevalent cytoplasmic localization is likely to result from increased nuclear export or decreased nuclear retention rather than an intrinsic defect in nuclear import, which would be expected to seriously impinge on their function.
The amino-acid changes in Rexb and Rexc do not affect the sequence of the nuclear localization signal (NLS) or the leucine-rich nuclear export signal/activation domain (NES/AD). However, the presence of 25 or 24 extra amino-acids in Rexb and Rexc respectively, immediately downstream of the NLS and 55 amino-acids upstream of the NES/AD (Figure 2) might induce conformational changes affecting the function of the NLS and/or the NES/AD. It is also possible that the extra residues affect the protein phosphorylation status, which is known to be important for Rex function , as well as subcellular localization in the case of Rex of HTLV-2 .
Distinct temporal regulation of mRNAs encoding Rex isoforms
These findings provide insight into the mechanisms controlling HTLV-1 expression and suggest that the production of alternatively spliced monocistronic mRNAs coding for Rex isoforms contributes to the two-phase kinetics of HTLV-1 expression. The resulting pattern of viral gene expression might be important to temporally restrain the expression of highly immunogenic viral epitopes (e.g. Tax, Gag, Env), thus favouring escape from the immune response and establishment of long term persistence in the host, a key feature of HTLV-1 infection. A better understanding of how these regulatory circuits are established and maintained will shed light on the basis of viral latency and may provide groundwork for the development of new therapies for eradicating persistent infections.
FR carried out the molecular cloning, immunofluorescence, immunoblot and functional assays, analyzed and interpreted the data, IC carried out the qRT-PCR assays, analyzed and interpreted the data. VA and VWV helped with in initial pilot experiments. VC and GF conceived the study, analyzed and interpreted the data, and DMD participated in its design and coordination and helped to interpret the results. All authors read and approved the final manuscript.
We thank Luigi Chieco-Bianchi for discussions and Charles Bangham and Graham Taylor for discussions and patient samples. The research was supported by investigator Grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC; nos. 4175 and 13378), an AIRC-Cariverona Regional Grant, and the University of Padua (Ateneo Grant no. CPDA124913/12).
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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- Gonçalves DU, Proietti FA, Ribas JG, Araújo MG, Pinheiro SR, Guedes AC et al (2010) Epidemiology, treatment, and prevention of human T-cell leukemia virus type 1-associated diseases. Clin Microbiol Rev 23(3):577–589PubMed CentralPubMedView ArticleGoogle Scholar
- Currer R, Van Duyne R, Jaworski E, Guendel I, Sampey G, Das R et al (2012) HTLV tax: a fascinating multifunctional co-regulator of viral and cellular pathways. Front Microbiol 3:406PubMed CentralPubMedView ArticleGoogle Scholar
- Nakano K, Watanabe T (2012) HTLV-1 Rex: the courier of viral messages making use of the host vehicle. Front Microbiol 3:330PubMed CentralPubMedView ArticleGoogle Scholar
- Hidaka M, Inoue J, Yoshida M, Seiki M (1988) Post-transcriptional regulator (rex) of HTLV-1 initiates expression of viral structural proteins but suppresses expression of regulatory proteins. EMBO J 7(2):519–523PubMed CentralPubMedGoogle Scholar
- Rende F, Cavallari I, Corradin A, Silic-Benussi M, Toulza F, Toffolo GM et al (2011) Kinetics and intracellular compartmentalization of HTLV-1 gene expression: nuclear retention of HBZ mRNA. Blood 117:4855–4859PubMedView ArticleGoogle Scholar
- Ciminale V, Rende F, Bertazzoni U, Romanelli MG (2014) HTLV-1 and HTLV-2: highly similar viruses with distinct oncogenic properties. Front Microbiol 5:398PubMed CentralPubMedView ArticleGoogle Scholar
- Cavallari I, Rende F, Bender C, Romanelli MG, D’Agostino DM, Ciminale V (2013) Fine tuning of the temporal expression of HTLV-1 and HTLV-2. Front Microbiol 4:235PubMed CentralPubMedView ArticleGoogle Scholar
- Corradin A, DI Camillo B, Rende F, Ciminale V, Toffolo GM, Cobelli C (2010) Retrovirus HTLV-1 gene circuit: a potential oscillator for eukaryotes. Pac Symp Biocomput 421–432Google Scholar
- Kimata JT, Wong FH, Wang JJ, Ratner L (1994) Construction and characterization of infectious human T-cell leukemia virus type 1 molecular clones. Virology 204(2):656–664PubMedView ArticleGoogle Scholar
- Schwartz S, Felber BK, Benko DM, Fenyö EM, Pavlakis GN (1990) Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1. J Virol 64(6):2519–2529PubMed CentralPubMedGoogle Scholar
- Ciminale V, Pavlakis GN, Derse D, Cunningham CP, Felber BK (1992) Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: novel mRNAs and proteins produced by HTLV type I. J Virol 66(3):1737–1745PubMed CentralPubMedGoogle Scholar
- Koralnik IJ, Gessain A, Klotman ME, Lo Monico A, Berneman ZN, Franchini G (1992) Protein isoforms encoded by the pX region of human T-cell leukemia/lymphotropic virus type I. Proc Natl Acad Sci USA 89(18):8813–8817PubMed CentralPubMedView ArticleGoogle Scholar
- Rende F, Cavallari I, Romanelli MG, Diani E, Bertazzoni U, Ciminale V (2012) Comparison of the genetic organization, expression strategies and oncogenic potential of HTLV-1 and HTLV-2. Leuk Res Treat 2012:876153Google Scholar
- Popovic M, Lange-Wantzin G, Sarin PS, Mann D, Gallo RC (1983) Transformation of human umbilical cord blood T cells by human T-cell leukemia/lymphoma virus. Proc Natl Acad Sci USA 80(17):5402–5406PubMed CentralPubMedView ArticleGoogle Scholar
- Hanon E, Hall S, Taylor GP, Saito M, Davis R, Tanaka Y et al (2000) Abundant tax protein expression in CD4+ T cells infected with human T-cell lymphotropic virus type I (HTLV-I) is prevented by cytotoxic T lymphocytes. Blood 95(4):1386–1392PubMedGoogle Scholar
- Bhat NK, Adachi Y, Samuel KP, Derse D (1993) HTLV-1 gene expression by defective proviruses in an infected T-cell line. Virology 196(1):15–24PubMedView ArticleGoogle Scholar
- Benko DM, Robinson R, Solomin L, Mellini M, Felber BK, Pavlakis GN (1990) Binding of trans-dominant mutant Rev protein of human immunodeficiency virus type 1 to the cis-acting Rev-responsive element does not affect the fate of viral mRNA. New Biol 2(12):1111–1122PubMedGoogle Scholar
- Schwartz S, Felber BK, Pavlakis GN (1992) Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein. J Virol 66(1):150–159PubMed CentralPubMedGoogle Scholar
- Kesic M, Doueiri R, Ward M, Semmes OJ, Green PL (2009) Phosphorylation regulates human T-cell leukemia virus type 1 Rex function. Retrovirology 6:105PubMed CentralPubMedView ArticleGoogle Scholar
- Narayan M, Younis I, D’Agostino DM, Green PL (2003) Functional domain structure of human T-cell leukemia virus type 2 rex. J Virol 77(23):12829–12840PubMed CentralPubMedView ArticleGoogle Scholar
- Cavallari I, Rende F, D’Agostino DM, Ciminale V (2011) Converging strategies in expression of human complex retroviruses. Viruses 3(8):1395–1414PubMed CentralPubMedView ArticleGoogle Scholar
- Cavallari I, Rende F, Ciminale V (2014) Quantitative analysis of human T-lymphotropic virus type 1 (HTLV-1) gene expression using nucleo-cytoplasmic fractionation and splice junction-specific real-time RT-PCR (qRT-PCR). Hum Retrovir Methods Protoc 1087:325–337View ArticleGoogle Scholar